Descriptive Study of a 488 Patients Cohort with Inherited Retinal Diseases and a Low Genetic Diagnosis Rate.

Backgorund: To analyze the distribution of inherited retinal diseases (IRDs), describe the clinical characteristics of patients, and determine the percentages of patients with genetic diagnoses in the Castilla and Leon region of Spain. Methods: All patients with an IRD seen in the two major referral units of Castilla y Leon during a 20-year period were included. The ages at symptom onset, diagnosis, and the last visit; sex; family history; history of consanguinity; type of inheritance; status of the fundus and electroretinogram; lens and macular status, visual acuity; and visual eld data were recorded. Patients were divided into those with retinitis pigmentosa (RP) and all others. The genetic mutations were gathered. Results: Four hundred eighty-eight patients with IRDs were studied, 216 with RP (of which 15.74% had syndromic disease) and 272 with other conditions (59.19% of which were macular dystrophies). A genetic study had been performed in 27.31% of patients (29.31% of which were negative) among the patients with RP and in 26.1% of the rest of the IRDs (28.16% of which were negative). In the group without a genetic diagnosis, 70.6% of subjects had good remaining vision. The mean delay in diagnosis was 6-16.2 years. Conclusions: The percentage of patients with IRDs that are candidates for undergoing personalized treatments including gene therapy in our region is low and must be improved. Most patients with RP who did not undergo a genetic study have good remaining vision, so they would be candidates for receiving treatments aimed at maintaining vision. There is a signicant delay in diagnosis.


Background
Inherited retinal diseases (IRD) are a group of rare Mendelian neurodegenerative conditions caused by mutations in genes that code for proteins essential for the functioning and maintenance of photoreceptors, retinal pigment epithelium and other retinal neurons leading to retinal dystrophies or optic neuropathies [1]. The prevalence rates vary between 1/750 and 1/5,000, depending on the region and level of consanguinity or ethnicity [2][3][4]. IRDs also are one of the main causes of irreversible blindness in young patients and have a highly signi cant impact on quality of life and health economics [5,6].
IRD can be classi ed as panretinal pigmentary retinopathies affecting primarily rods or cones in which pigmentary clumping occurs secondary to photoreceptor death; macular dystrophies with only central retinal involvement; stationary conditions in which the photoreceptors do not function but do not die; optic nerve disease primarily due to involvement of ganglion cells; and other less frequent diseases such as vitreoretinopathies [7,8]. The most frequent IDR is retinitis pigmentosa (RP) that usually is an isolated ocular condition, but 20-30% of cases may be syndromic [2,9,10].
The inheritance patterns of these pathologies are usually monogenic, autosomal dominant (AD), autosomal recessive (AR), or X-linked recessive (XL) but on rare occasions can be digenic, mitochondrial, or have another pattern [2]. More than 3,000 mutations have been identi ed in more than 300 genes in syndromic and non-syndromic diseases, and that number is constantly increasing thanks to greater access to next-generation sequencing (NGS) [5,11].
Creation of organized and shared networks to create registries and biobanks would facilitate the performance of epidemiologic studies and identify the causal mutations using exome and whole genome sequencing provided by the NGS techniques, which has become an endeavor of the highest priority [5].
This advancement is crucial to elucidate the molecular pathologies of disease, diagnose unresolved cases, improve genetic counseling, and develop new advanced therapies including gene therapy, all of which will change the paradigm of IRDs, as new treatments may improve vision and prevent blindness [12]. Thus, identifying the genes involved and the clinical status of our patients will help identify patients who are candidates for treatments based on personalized medicine or involvement in one of the more than 300 ongoing clinical trials for this group of diseases [13].
The objective of the current study was to describe the relative frequencies of different diseases with the clinical patterns of various subtypes of monogenic retinal degenerations and estimate the molecular diagnostic success rates in describing the causative genes in a Spanish region. This study will increase the knowledge about these dystrophies in our population and provide information to investigators and the

Patients
The RHUH database included 82 patients with RP seen between 1995 and 2019. The IOBA database included 518 consecutive patients with a diagnosis of an IRD seen between 1998 and 2019. All double entries in both units and cases with a doubtful diagnosis (those cases without clear clinical ndings or genetically con rmed diagnosis) or an incomplete dataset were excluded.

Data collection
For the RP group, the variables studied included sex; ages at symptom onset, diagnosis, and the last visit; family history; lens status; inheritance pattern, and genetic testing data. The electrophysiology status was recorded as normal, diminished, or abolished for the scotopic and photopic responses. The ocular fundus was classi ed as having a classic pattern in the spicules, scarce pigment, granular pattern, atrophy, perivenous pattern, and sector RP. The macular status was classi ed as preserved, atrophic, or edematous based on ocular fundus, auto uorescence and Spectral Domain Optical Coherence Tomography. The best-corrected visual acuity (BCVA) was recorded using a Snellen chart and converted to the logarithm of the minimum angle of resolution (logMAR) using a validated procedure [14]. The BCVA was collected for each decade of life for each eye; for analysis, the patients were divided into ve groups according to the low vision scale of the World Health Organization based on the BCVA in the eye with the better vision, i.e., no low vision (LV) (logMAR < 0.20), mild LV (logMAR > 0.20-0.50), moderate LV (> 0.50 and < 1.00), severe LV (1.00-1.30), and blindness (> 1.30). The visual eld (VF) data were divided into ve groups: central VF < 5 º ; central VF between 5º and 10º; central VF > 10º; total abolition of the VF; and central rather than peripheral involvement.
For the remaining IRD patients, we recorded the age; sex; baseline BCVA; ages at symptom onset and diagnosis; inheritance pattern, and mutated gene if available.

Statistical analysis
For the quantitative variables, the descriptive statistics used were the mean, standard deviation (SD), and the range (maximal and minimal values). These values were calculated in Excel tables and with the SPSS program version 24 for Windows (IBM Corp., Armonk, NY, USA) for 1989-2017. For the qualitative variables, the numbers and percentages of each category were used.
The Student's t-test was used to compare the means between two groups for quantitative variables, and the chi 2 test for qualitative variables through a contingency table or Fisher's exact test if the expected frequencies were small. P < 0.05 was considered signi cant. The analyses were performed with the statistical package SPSS, version 24.

Results
The clinical records of 600 patients were reviewed with suspected IRD, and the hereditary cause could only be clinically con rmed in 488 patients. Of the RP subgroup, 112 (51.85%) were men and 104 (48.15%) were women. Thirty patients (13.9%) had Usher syndrome (7 type I, 21 type II, and 2 type III). Two patients had Bardet-Biedl syndrome, one retinal degeneration related to Sjögren-Larsson syndrome and one to Crouzon syndrome. The inheritance pattern could not be established in 28 patients due to lack of information. Of the remaining 194 patients, 47 (24.23%) were classi ed as sporadic cases, 104 (53.61%) with AR, 29 (14.95%) as AD, 13 (6.7%) as XL, and one (0.52%) with dominant XL that may also be a severe female carrier. In the syndromic RP group, 19 patients (66.33%) were AR and eight (26.66%) were sporadic. The family history was positive in 105 (48.61%) patients of the total RP sample. Twenty-one (11.11%) of 189 patients were children of consanguineous parents and 33 (17.46%) of "relative" consanguineous parents (both born in a village with less than 500 inhabitants).
The mean age of symptom onset in patients with RP was 17.30 ± 15.27 years (range, 1-73) and the mean age at diagnosis was 31.48 ± 17.68 years (range, 2-82) years, indicating an average delay in diagnosis of 16.21 years. No signi cant differences were found between the mean ages of syndromic RP and non-syndromic RP or between the different patterns of inheritance.
The mean age of patients with RP at the last visit was 47.96 ± 17.26 years (range; 3-89). Information about the lens was obtained in 208 patients on their last visit. Cataract developed at a mean age of 42.44 ± 15.84 years (range, 2-86), and 65 (30.09%) were pseudophakic. The mean age at which cataract surgery was performed in the overall sample was 48.30 ± 12.01 years (range, . No signi cant differences were found between syndromic and non-syndromic RP or between different inheritance patterns. The macular status was recorded in 211 patients with RP at their last visit and was preserved in 74  (Table 1), although 11 of the remaining 17 patients who were negative had been tested before 2013 before implementation of the NGS techniques. Interestingly, in the group without a genetic diagnosis, 70.6% of subjects had useful remaining vision.
Other inherited retinal dystrophies Concerning IRD other than RP the frequency distribution of each disease, distribution by sex, mean age at symptom onset, and mean age at diagnosis are presented in Table 2. The delay in diagnosis for progressive cone dystrophy and cone-rod dystrophy was 10 years; in central areolar choroidal dystrophy (CACD) 14 years; in Sorsby's fundus dystrophy 7 years; in Best's vitelliform macular dystrophy 8 years; in congenital stationary night blindness 11 years; in congenital achromatopsia 6 years, and in dominant optic atrophy 15 years. The inheritance patterns are presented in Table 3.    Only 71 (26.1%) of the 272 patients diagnosed with an IRD other than RP had a genetic diagnosis, but it was negative in 20 (28.16%). The results are presented in Table 1.
A comparison of our data with results obtained by other research groups is presented in Table 4.

Discussion
The current study presents interesting data about the relative frequencies of inherited retinal diseases and their clinical appearance, inheritance patterns, age at diagnosis, and results of genetic testing in a Spanish region with 2,500,000 inhabitants.
As expected, RP was the most frequently inherited retinal disease found in the current study. The RP frequencies vary little between published studies and range from 34.75-43.06% [8,15,16]. Even the group of syndromic RPs as a whole had similar frequencies (Table 4). Usher syndrome was the most frequent form of syndromic pigmentary retinopathy as reported previously [8,15,16]. Regarding macular dystrophies, Stargardt's disease was the most frequently seen entity in the current study and in most studies but with important variations ranging from 5.51% reported by Bocquet et al. in Southern France to 20.62% reported by Motta et al. in Brazil [8,15]. The reported frequencies of other macular dystrophies vary widely among studies of different populations, and the current study has the higher percentage [8,15,16]. Such wide variations can be due to several factors: it is unclear if some macular dystrophies included in the current study also were included in other studies; the de nite diagnosis of a particular macular dystrophy can be challenged; moreover, geographic variations in the frequency of some mutations can promote geographic disparities in the frequency of some entities as happens with the higher rate of CACD in the Spanish population compared with others [17,18]. Although consanguinity can play an important role in the prevalence of IRD in some populations [19], the current study had consanguinity data, including relative consanguinity data (11.11% and 17.46%, respectively), similar to those published in most populations [15,20].
Concerning the inheritance patterns in the RP group, the rate of AR inheritance in the current sample was high (53.61% vs. 16.0%-39% in other studies), while sporadic cases occurred less frequently than in other studies (24.23% vs. 40%-51% in other studies) [4,[21][22][23][24]. Nevertheless, 28 patients in our sample did not provide family history data and thus they were not included in this analysis; if they had been considered as sporadic cases, this gure would have approached 50%.
Within the RP group, the ERG was abolished or diminished in most patients, similar to data reported by Berson et al [20,25].
With small variations, the clinical presentations of IRDs in the current sample was similar to those already described. Regarding the ocular fundus, most had the classic pattern of pigmentation in the spicules. The percentage of cataract patients (39.42%) was similar to the data provided by Testa et al [24]. and slightly lower than in other studies (45%-52%) [26]; the group of pseudophakic patients among the current cases (31.25%) was greater than that found in the other studies (15.4%-20%). [10,[27][28][29] The mean age at the time of cataract surgery was similar to those reported previously [27][28][29]. Finally, the prevalence of macular edema in our RP sample (17.06%) was lower than in other studies (23%-50%) [10,[30][31], although it was similar to that reported by Hirakawa et al [32]. Genetic diagnosis of IRDs is desirable for many reasons because it can provide a de nitive diagnosis, which can deliver great relief to patients and families; it also helps physicians to better de ne the inheritance pattern and the risk for the remaining family members, and it is crucial to identify patients for potential enrollment in clinical trials or new advanced therapies. Besides, 71.6% of the patients in the current series without a genetic study had good remaining vision, so they could be candidates for gene therapy. The rst important di culty for genetic diagnosis of IRDs is that the clinical diagnosis of different phenotypes may be due to the same mutation and vice versa [33]. Thus, the wide variety of IRDs and their relative low frequency make it di cult for the general ophthalmologist and the retinal specialist to establish an accurate diagnose and focus the search on one or a small number of gene mutations. Another important factor is the possibility for ophthalmologists and patients to access a clinical genetic unit. In our case, Castilla y Leon region lacks a genetic unit. These reasons explain the low rates of genetic diagnoses obtained in the current study (22.95%), which was slightly lower than those published in other such studies (30.11% and 32.10%) [8,15].
Delayed diagnoses are common problems in IRDs and may result from deferral of patients with referring symptoms and/or the inexperience of doctors who rst treated them. Data from the Survey of the Delay in Diagnosis for Rare Diseases in Europe con rm that this delay generally occurs in the diagnosis of any rare disease [34]. Moreover, late diagnosis of stable diseases may be due in part to their moderate visual impairment and minimal fundus changes in children who do not complain [35]. Entities with a more characteristic phenotype can be recognized early, but in many cases a conclusive diagnosis of an IRD cannot be made until several visits and electrophysiologic and imaging evaluations are performed and interpreted correctly.
The major strength of the current study was that the patients were part of ordinary specialized practices and the sample size was adequate considering that the diseases are rare. It also emphasizes the need to improve access to genetic testing in our population.
But the current study has important limitations. First, the sample size of each disease was small except for that of RP. However, these are rare diseases and, in this context, relevant ndings could be highlighted. Another constraint was the heterogeneity in the follow-up of the patients (some, up to 20 years of followup and others, only infrequent visits). In addition, the presence of macular edema may have been underestimated, especially in patients with few visits during the rst years in which optical coherence tomography was unavailable, although this happened only in a small number of patients. A major problem was that the vast majority of patients do not have a molecular diagnosis and the state-of-the -art in IRD is based on both clinical and molecular diagnosis. In this context comments about inheritance patterns must be taken with caution. Finally, many of the genetic studies with negative results were very old and patients need to be reassured using NGS techniques. Nevertheless, this is something we wanted to point out to be able to ask regional management for a change in their policy denying test to this group of patients and families.
Knowing the rate of genetic testing in actual clinical practice can be a good starting point to plan future actions such as genetic studies with new diagnostic techniques for as many subjects as possible, which would allow classi cation of the pathology from the genotypic point of view in our population, to move forward in the search for new mutations and properly diagnose patients with doubtful diagnoses.

Conclusions
Only 27.31% of the patients with RP and 26.1% of the rest of the IRDs had undergone genetic testing, but in more than two thirds (71.31%) of those with RP the test results were positive and facilitated the identi cation of more causative mutations and genes in our population. Most RP patients (71.6%) had residual vision, which allows them to be candidates for treatments aimed at preserving their vision, and we need to promote their prompt genetic diagnosis. The current study con rmed the substantial delays in diagnosing most of these diseases and the need to perform more genetic studies in our region what also worries us. By highlighting our weaknesses we want to move regional managers to improve these data.

Declarations
Ethics approval and consent to participate . Administrative permissions were acquired by our team to access the data used in our research.
MDA wrote the nal manuscript. RMC, MRS and HST revised critically the work. All authors read and approved the nal manuscript.